Nematodes of the genus Trichinella Railliet, 1895 (Nematoda: Trichinellidae) are widespread zoonotic pathogens that affect a wide range of hosts, from domestic and wild animals to humans (1). Currently, ten species and three genotypes of Trichinella are known, separated into two clades, one which includes species capable of modifying myocytes into nurse cells and encapsulating themselves in the muscle tissue of the hosts, and the second which includes species incapable of this tissue modification (1, 2). Although the genus Trichinella has been known and studied since its description in 1835, knowledge about the genus is still dynamic, with a new species described in Canada in 2020 from samples of a wolverine (Gulo gulo), a wild mustelid common in North America (1, 3).

Several hosts, such as wild carnivores, rodents, wild boars, birds and reptiles, have already been related to the epidemiological chain of trichinellosis (4, 5). The cycle is divided into two categories: a domestic cycle, where domestic pigs are the main reservoirs, and a sylvatic cycle, which has wild boars and wild carnivores as the main hosts of the nematode (6). Noteworthy are the feeding habits of carnivores, which includes rodents species, which are also an important host for the parasite (7). Transmission occurs through ingestion of muscle tissue infected with larvae, and behaviors such as predation, cannibalism and necrophagy are closely related to infection in animals. In humans, the infection is mainly associated with the consumption of raw or undercooked pork and game meat, also without veterinary inspection (1, 8, 9).

In South America, the parasite has been detected in wild animals such as pumas (Puma concolor), opossums (Didelphis albiventris), wild boars (Sus scrofa), armadillos (Chaetophractus villosus) and sea lions (Otaria flavescens) in Bolivia, Argentina, Ecuador and Chile (7, 10). In Brazil there is only serological evidence that the agent is circulating in wild boars (11). In contrast, Argentina reports a high number of human infections, with more than 6,000 cases reported between 2012 and 2018 (12). Also, Chile e Bolivia reported cases in humans by the detection of anti-Trichinella antibodies (12, 13). However, studies in other South American countries are scarce or absent, hindering the proper comprehension of the epidemiology of human trichinellosis in this region.

Considering the importance of wild carnivores in the epidemiology of trichinellosis, this study aimed to investigate Trichinella spp. infection in free-living wild carnivores in the state of São Paulo, Brazil.

No Trichinella spp. larvae were found in this study, either by artificial digestion or histopathological analysis. To date, there has been no record of diagnosis of nematode larvae in any study carried out in Brazil, whether in domestic or wild animals. More than 15,000 domestic pigs had samples analyzed by AD in the state of Paraná between 2002 and 2011, all of which were negative (19–21). Furthermore, 14,852 horses were tested in the states of Minas Gerais, Goiás, and Bahia between 2014 and 2016, with no observation of the nematode (22). In addition, 594 rats were tested in the region of Santos, São Paulo and not a single sample was positive (23). Only a few published studies have tested for trichinellosis in wild animals using artificial digestion and/or other diagnostic methods in Brazil. Between 2018 and 2020, 15 wild carnivores (05 Puma concolor, 03 Leopardus pardalis, 02 Chrysocyon brachyurus, 04 Cerdocyon thous e 01 Leopardus guttulus) were tested in São Paulo state (11), while 71 other wild animals (29 Didelphis albiventris, 11 Nasua nasua, 10 Cerdocyon thous, 07 Dasypus novemcinctus, 06 Leopardus guttulus, 06 Sphiggurus spinosus and 02 Puma concolor) run over in the northern region of Paraná, were analyzed from 2016 to 2021 (24). The data from the present study add further evidence of the absence of circulation of the agent in wild carnivores in Brazil, despite serological evidence of infection by Trichinella spp. in wild boar (11).

Countries near Brazil, such as Chile and Argentina, have recorded the presence of Trichinella spp larvae. (T. spiralis, T. patagoniensis, T. pseudospiralis e T. britovi) in wild carnivores, such as the guigna (Leopardus guigna), lesser grison (Galictis cuja) and jaguar (Puma concolor), using the diagnostic method of AD (9, 12). Due to its high sensitivity, this technique is considered the gold standard for diagnosing Trichinella spp. by the World Organization for Animal Health (WOAH) (25).

The health of wild animals is known to be related to human health given their importance as carriers or reservoirs of zoonotic pathogens (26, 27). Currently, the transmission of trichinellosis is more related to the consumption of undercooked meat from wild animals than from domestic pigs due to technological advances in pork farms (28). However, international trade requires testing for Trichinella spp. in pig production in countries where the disease occurs, increasing the cost of importing products (2). Additionally, subsistence-type farms with low confinement as well as free-range or organic pig production increase the risk of exposure to Trichinella and many outbreaks in Easterns Europe and Argentina are related to this type of production (11, 29).

Despite the negative results obtained using the artificial digestion method, a Brazilian government agency reported 24 positive cases for Trichinella spp. out of 554 wild boars tested using the ELISA technique (IDEXX xChek^®) between 2012 and 2016 (12). The samples were obtained from wild boar slaughtered in the states of São Paulo, Mato Grosso, Mato Grosso do Sul, Rio Grande do Sul, and Santa Catarina, resulting in the change of Brazil’s health status for Trichinella from “never reported” to “restricted in some areas” (30). Additionally, 07 wild boars out of 115 animals with serum tested by indirect ELISA in São Paulo state from 2018 to 2020 were also positive for trichinellosis (11). The use of indirect diagnostic methods such as ELISA can be an essential tool in the surveillance of zoonotic diseases such as trichinellosis in wild animals. However, the validation of serological assays for several wild animals is hindered by the lack of reference sera (31) and due to the numerous parasitic, bacterial, fungal and viral agents that can affect these animals, the chances of cross-reactions and false positives are high (32). Another factor that makes it difficult to carry out serologies on the carcasses of animals that have been road killed is the coagulation and autolysis of the blood. Direct methods are more suitable for diagnosing Trichinella spp. in these cases. Furthermore, using the carcasses of road killed animals is a non-invasive way of performing epidemiological surveillance of several pathogens, maximizing the use of biological samples and reducing the risk to both humans and animals.

Microscopy showed in 14% (20/142) of the animals (07 Puma concolor, 04 Cerdocyon thous, 03 Leopardus pardalis, 03 Lontra longicaulis, 01 Nasua nasua, 01 Chrysocyon brachyurus, 01 Leopardus guttulus), rounded, basophilic and encapsulated structures in the striated muscle cells, compatible with Sarcocystidae protozoan cysts. The protozoan cysts visualized under microscopy, even if they were accidental findings, show the effectiveness of the technique in visualizing intracellular parasites in muscles when present. As observed in a study in 2011 in Texas, USA, using the diagnostic methods of histology and artificial digestion of the tongues of 77 coyotes (Canis latrans) to test for Trichinella spp., one animal tested positive only by histology. In addition to being in agreement with AD, offering greater accuracy in the result (33). Therefore, it constitutes an auxiliary technique for the diagnosis of trichinosis. In 2003, researchers in Chile diagnosed trichinosis infection in an Inca human mummy from around 1500 AD, using histological techniques and indirect immunofluorescence (34). Despite not being the gold standard for diagnosing this nematode, histopathology is an alternative for confirming infection and diagnosis.

Wild predators are associated with the maintenance of Trichinella in the wild cycle of all disease sites in the world (28). The meat consumption from these animals is cultural in some Latin American countries, such as Chile. Although hunting wild carnivores is illegal in Chile, researchers confirmed the presence of Trichinella spiralis in a semi-cured carcass of a jaguar (Puma concolor) that was slaughtered for consumption (35). Also prohibited in Brazil, this practice is considered cultural in some regions (36, 37). Carnivores such as the ocelot (L. pardalis), oncilla (L. guttulus), Jaguarundi (H. yagouaroundi) and crab-eating fox (C. thous) have already been reported in studies as a source of protein for consumption and/or medicinal use (38, 39).

Access to samples of wild carnivores, especially endangered animals protected by law, can be hampered by various factors. Therefore, the necropsy of carcasses of animals that have already died, as well as not interfering with conservation or causing stress to the animals, provides us with a lot of information (40). It is possible to observe places of occurrence, points of greatest trampling, and evaluate stomach contents, among other ways of using them for educational purposes. It is also possible to monitor numerous diseases of human and animal importance.

The sensitivity of the AD diagnostic is theoretically 1 larva per gram of muscle. However, due to the non-uniform larvae distribution in the tissues and limitations of the technique, the current sensitivity is considered 3 to 5 larvae per gram for 1g of muscle (41, 42). However, it is not known whether this method has the same sensitivity for detecting pre-encapsulated larvae (43). With regard to species from the non-encapsulated clade, studies show that the sensitivity of AD is between 7 and 17 larvae per gram (44). Therefore, factors such as the distribution of larvae in the muscles of carnivores and low parasite rates can lead to false negatives. Samples freezing may reduce the sensitivity of the technique, especially in the case of larvae that are not resistant to freezing, and therefore the tissues should ideally be maintained at 2-8°C (45). However, due to the logistics of the distance between the laboratory and the institutions that collected the carcasses, freezing is the best alternative of accessing a greater number and diversity of carnivore species.

The epidemiological surveillance of trichinellosis in wild boars is already being carried out in Brazil, as these animals are important hosts for the nematode (46). Wild animals, especially carnivores, are natural hosts of Trichinella spp. and the parasite develops better in these animals than in domestic animals (39). Given the way trichinellosis is transmitted and the fact that these animals are at the top of the food chain, epidemiological surveillance of wild carnivores and wild boars is essential to understanding the epidemiology of the disease in Brazil.

To date, there is no direct evidence of Trichinella spp. nematodes circulating in wild carnivores in São Paulo state